Protocol detail

Protein Extraction from Transduced Cells

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Protein extraction from transduced cells was performed using Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA)^{49 (link)}. Samples were sonicated for 15 s at 10 mA, followed by centrifugation at 16,000 RCF for 10 min at 4 °C, and transferal of supernatant to a new tube. Samples were quantified with the DC Protein Assay (Bio-Rad, Hercules, CA) using the recommended protocol. Sample absorbance at 750 nm was quantified using the PowerWave XS2 Microplate Spectrophotometer (BioTek, Winooski, VT).

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Moses C., Hodgetts S.I., Nugent F., Ben-Ary G., Park K.K., Blancafort P, & Harvey A.R. (2020). Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing. Scientific Reports, 10, 11393.

Publication 2020

Cell Lysis Buffer DC Protein Assay PowerWave XS2 Microplate Spectrophotometer

Assay Buffer Cell Centrifugation Protein

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Variable analysis

independent variables

Cell lysis buffer used for protein extraction

dependent variables

Protein concentration in samples

control variables

Sonication duration (15 s)
Sonication amplitude (10 mA)
Centrifugation speed (16,000 RCF)
Centrifugation duration (10 min)
Centrifugation temperature (4 °C)

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